To date, our best tools for assessing the frequency and affinity of CD4+ T cells have been peptide:MHC (pMHC) tetramers or functional assays; neither of which effectively identify all of the responding cells to a particular antigen. This is in contrast to CD8+ T cells where these techniques prove more accurate. Generally, pMHC tetramers for class II antigens are difficult to produce and identify a small percentage of responding CD4+ T cells especially when antigen is derived from self proteins. The reason for this is that tetramer is based on affinity and if affinity is too low, then the tetramer is of limited use in assessing the response Functional responses also underestimate the number of antigen reactive CD4+ T cells. In the case of responses directed against myelin antigens for instance, the effector cytokine response is often determined using strong pharmacologic agents such as PMA and ionomycin, which hammer the T cell signaling cascade and may have little relevance to the cytokines being produced in response to antigen itself. To better assess the range of responding CD4+ T cells, we have begun to use micropipette based affinity assay. We report here a major advancement in assessment in the frequency and affinity of CD4+ T cells directed against myelin or viral antigens. Therefore for the first time, it is possible to track the range of affinities of a CD4+ T cells response during disease progression. Our preliminary findings have led to the following central hypothesis that the CD4+ T cell affinity profile directly impacts disease outcome and immune mediated tissue damage in the CNS. Three specific aims are proposed to test this hypothesis focused on CD4+ T cells specific for self antigen myelin oligodendrocyte glycoprotein and pathogen associated lymphocytic choriomeningitis virus antigens. Aim 1- Identify the affinity of CD4+ T cells over the course of chronic autoimmune disease and viral infection. Aim 2- Define the connection between T cell affinity and effector phenotype. Aim 3- Establish the contribution of high versus low affinity T cells in response to antigens found in the CNS.